presentasi biokim ii

7/29/2019 Presentasi Biokim II

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INTRODUCTION

Muscle and whole body maximal aerobic performancedecline with age in humans.

The loss of muscle mass is an important contributor tothis decline but the role of oxidative capacity per musclemass is less clear.

Age related changes in volume specific oxidative

capacity have been inferred from both in vitro and in vivomeasurements of muscle oxidative properties.


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Two tools havethe potential for

allowing a

determination ofmuscle oxidativecapacity in vivo.

Magnetic resonance(MR) methods that

make possiblenoninvasive

assessment of the

change in muscleenergetics in vivo.

The second toolallowing us to

determine oxidativecapacity in muscle is

the model of the

control of oxidativephosphorylation


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PURPOSE

The purpose of this study was to determine theoxidative capacity of muscle and how it differs

between adult and elderly groups.


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METHODS


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Subject

An adult group consisted of nine subjects (6 males, 3 females) ranging inage from 25 to 48 years (388 79 years, mean s.d.).

An elderly group consisted of 40 subjects (18 male, 22 female) ranging in

age from 65 to 80 years (688 59 years). Body mass was not significantly different between the two groups (adult

698 28 kg, elderly 721 22 kg, means s.e.m.).

Subjects were not involved in a formal exercise training programme, were ingood health and had no significant cardiac, neurological or musculoskeletal

disease. Nine of the elderly female subjects were receiving hormone replacement

therapy.

Subjects activity profilesranged from housework, yardwork, and occasionalwalks to aerobic activities several times per week.

All subjects voluntarily gave informed, written consent.


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Experimental Protocol

Using the dynamics of [PCr] and pH during stimulation to estimateglycolytic + production and during aerobic recovery from exerciseto measure ATP supply and estimate oxidative capacity. Spectraldata were collected at 6 s intervals over an 8 min period using thefollowing protocol.

Control period (60 s, 10 spectra): baseline data were obtainedduring resting conditions to establish initial metabolite peak areasand pH under partially saturating nuclear MR data acquisitionconditions.

Stimulation period (120 s, 20 spectra): a 3 Hz electrical stimulationperiod was used to decrease [PCr]. Glycolytic + production wasdetermined from the [PCr] and pH changes as previously reported

Aerobic recovery (300 s, 50 spectra): upon cessation of stimulation,the extent and time course of the aerobic PCr recovery was

followed and used as the basis of the oxidative phosphorylationdeterminations.


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Muscle Stimulation

activated thequadricepsmuscles by

transcutaneouselectrical

stimulation of thefemoral nerve

used EMG to

monitor muscleactivation andestablish the

maximalstimulating

voltage.

The activeelectrode wasplaced over the

belly of the vastuslateralis muscle

determine theintensity thatevoked themaximum

EMGresponse foreach subject

stimulations ofsupramaximalintensity (13

15 timesmaximal) weredelivered for 2min at 3 Hz.


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Magnetic ResonanceDeterminations A 9 cm diameter surface coil tuned to the phosphorus

frequency (259 MHz) was placed over the vastus lateralismuscle of the thigh.

The B1 field homogeneity was optimized by offresonanceproton shimming on the muscle water peak.

The unfiltered PCr linewidth (full width at halfmaximal height)was typically 48 Hz.

Each subject had a high resolution control 31P MR spectrumof the resting muscle taken under conditions of fully relaxednuclear spins (16 freeinduction decays (FID) with a 16 s

interpulse delay) using a spectral width of 1250 Hz and 2048data points. Measurement of changes in [PCr], [ATP], [P] and pH during

and following stimulation were made using a standard 1 pulseexperiment with partially saturated nuclear spins (15 sinterpulse delay).


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Calculations

The linear model of oxidative phosphorylation was used.

The first step in using this model is to fit the recovery of [PCr] from exercise to the restinglevel using a monoexponential equation taken from a resistancecapacitance (RC)circuit:

[PCr]t = [PCr]0 + [PCr] (1 exp(t kPCr)), (1)

where the levels at time t and the beginning of recovery are [PCr]t and [PCr],

respectively; kPCr is the rate constant of PCr recovery (kPCr = 1time constant ())and [PCr] = [PCr]rest [PCr].

The initial rate of change of PCr is given by the following derivative of eqn (1):

d[PCr]/dt = kPCr[PCr] exp(t kPCr). (2)

The instantaneous rate at t = 0 reduces the equation to:

d[PCr]/dt = kPCr[PCr]. (3)

This equation predicts the oxidative phosphorylation rate (d[PCr]dt) at any given [PCr]based on the characteristic kPCr of each muscle.

To estimate oxidative capacity, we assumed that [PCr]rest reflects the maximum range ofchange in [PCr] (i.e. [PCr]max = [PCr]rest 0).

Thus, an estimate of the oxidative capacity of the muscle comes from the characteristickPCr of that muscle and [PCr]rest in eqn (3).


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Oxidative capacity per mitochondrial volume

The average oxidative capacity of mitochondria hasbeen measured as the whole body maximum oxygenconsumption divided by the total mitochondrial volumeof the musculature.

This method yielded a oxidative capacity of between 4and 5 ml 2

1(ml mitochondria)1, which averages27 mol ATP 1 (ml mitochondria )1 for a P/2(phosphorylation to oxidation ratio of mitochondrial

respiration) of 6.


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Conts

An independent estimate of the maximal rate of mitochondrialrespiration comes from isolated mitochondrial studies .

Since muscle oxidizes >85% pyruvate at the maximal oxygenuptake rate, the substrate pair best suited for comparison with invivo mitochondria is pyruvate and malate.

This substrate pair yielded a maximum oxidative rate of 17 molATP 1(ml mitochondria)1 (2.7 ml 2 1( ml mitochondria

)1) at 30 C, when corrected for a P/2 of 6 and 0.7 ml 2O per mltissue.

Correcting the rate to 37 C (assuming a doubling of rate with each10 C, i.e. 10 = 2) yields 27 mol ATP

1(ml mitochondria)1

Thus isolated mitochondria and in vivo mitochondria have the samemaximum oxidative rate per mitochondrial volume. This value is used to estimate muscle oxidative capacity based on

mitochondrial volume density.


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Glycolisis

The glycolytic H generated was quantified from thechange in PCr and pH through stimulation:

+= pHtot + (-)PCr, (4)

where pH and PCr are the changes in pH and PCrduring exercise, tot is the buffering capacity of theindividuals muscle, and is the proton stoichiometriccoefficient of the coupled Lohman reaction

Glycolytic ATP synthesis is related to H production bythe ATP/ + stoichiometry of glycogenolysis andglycolysis


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Tissue Analysis

used the Bergstrom needle biopsy technique to acquiretissue from the midthigh level of the right vastus lateralismuscle.

A 25 mg piece was immersion fixed in glutaraldehyde,processed for electron microscopy, andmorphometrically analysed

Any remaining tissue was freezeclamped immediatelyaftercollection and stored at 80C prior to HPLCmetabolite analysis

Metabolite concentrations were expressed per volume of cellwater by assuming 07 ml intracellular water (g muscle

mass))1 as found for human muscle biopsy samples


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Statistics

Using Students twotailed paired and unpairedt tests to determine differences between

groups and standard linear regressionmethods for analysis of correlations.

Statistical significance was defined at the 0.05

level.


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RESULTS

three steps to achieve the goal ofevaluating the change in oxidative

capacity with age.

MR

spectroscopy was used to quantifythe metabolite levels in

resting muscle, during stimulationand through recovery.

the PCr recovery from exercise toestimate

the muscle oxidative capacity

evaluation thecontribution of a loss of

mitochondrial volume density and

mitochondrial function to thechange in oxidative capacity

with age.


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Metabolite levels and dynamics

The metabolite levels quantify using acombination of HPLC analysis of muscle

biopsies and the peak areas in the MR spectra


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Resting levels

Figure 1. Stack plot of every third 31P NMR spectrum during rest, stimulation and recovery inan elderly individual

The abscissal scale references PCrto a chemical shift () of 254 p.p.m. P, inorganicphosphate.


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Table 1. Metabolite levels determined by HPLC from musclebiopsies of the vastus lateralis and byMR of the same muscleMetabolite Adult Elderly Literature[ATP] (mM) 68 06 (3) 59 02 (32) 82Total [Cr] (mM) 434 52 (2) 467 11 (31) 42[PCr] (Mm) 267 35 (3) 260 10 (32) 32

[PCr]/[ATP] 430 02 (32) 39MR [PCr]/[ATP] 400 011 (9) 453 010 (38) * MR [P]/[ATP] 052 003 (9) 062 003 (38) MR [PDE]/[ATP] 075 014 (9) 076 005 (38)

Values are means s.e.m. with the sample size given inparentheses. Literature values are from Harris et al. (1974) forsubjects 1830 years old. PDE, phosphodiester. * Significantdifference between adult and elderly groups.


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Dynamic

Figure 2. Metabolite levels as a function of time during the stimulation andrecovery experiment Symbols indicate means and error bars indicates.e.m. Doubleheaded arrows indicate the duration of stimulation. adultsubjects; elderly subject


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Table 2. Metabolite levels determined by MR at rest and at the end ofstimulationQuantity Group Rest Stimulation end Stimulation Rest[PCr] (mM) Adult 2924 160 2044 132 880 137 *

Elderly 2711 088 1824 079 887 038 *[P1] (mM) Adult 432 069 1162 178 730 132 *

Elderly 387 022 1112 074 724 056 *Ph Adult 7063 0011 7040 0016 0022 0022

Elderly 7058 0006 7020 0009 0036 0011 *[ADP] (mM) Adult 0030 0004 0062 0007 0032 0005 *

Elderly 0031 00015 0065 0002 0033 0002 *Values are means s.e.m. The sample size was 9 for the adult group and 40for the elderly group. Significant difference between conditions.


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Oxidative phosphorylation rate

Next step was to determine the oxidativephosphorylation rate and estimate the oxidative

capacity. Which compared the initial rate of oxidative

phosphorylation estimated from themonoexponential fit of the PCr recovery to thatmeasured directly.


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Figure 3. [PCr] recovery following stimulation Continuous lines aremonoexponential fits to the data for an adult and an elderly subject.The vertical dashed lines denote the time constant for each recovery.


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Figure 4. [PCr] recovery rate constant (kPCr) as a function of age The linerepresents the regression equation:

y = 00005x + 0057 (2= 035)


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Figure 5. Oxidative capacity as a function of age The line represents the regressionequation: y = 0017x + 1777 (2= 034)


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Oxidative and mitochondrial properties vs.age

The final step was to evaluate the structural basis for the decline inoxidative capacity with age by determining the mitochondrial volumedensity (VV(mt,f)) from biopsies taken at the same site as the MR

determinations. These results confirm the lower oxidative capacity per mitochondrial

volume for the elderly vs. adult muscle shown in Fig. 6.


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Oxidative and mitochondrial properties vs.age

Figure 6. Oxidative capacity, mitochondrial volume density (VV(mt,f)) and oxidativecapacity/VV(mt,f) in the adult and elderly groups

Values are means s.e.m. and asterisks denote significant difference from the


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DISCUSSION

Oxidative recovery

Muscle vs. mitochondrial oxidative capacity

Adult vs. elderly oxidative capacity


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Oxidative recovery

A clear indication of the change in oxidative properties betweenadult and elderly muscle is the twofold difference in the time courseof PCr recovery from exercise shown in Fig. 3

Two results support the notion that kPCr is a characteristic of theoxidative properties of mitochondria in muscle.

First, kPCr is independent of [PCr] to 50% PCr depletion in anumber of rodent and human muscles . Only at low pH levels doeskPCr vary in a given muscle, because [H] significantly affects thecreatine kinase equilibrium thereby altering the link between [PCr]and oxidative phosphorylation.

Second, kPCr is proportional to oxidative enzyme activity in bothhuman and animal muscle, which indicates that kPCr is determinedby the mitochondrial properties of he muscle.


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Muscle vs. mitochondrial oxidativecapacity

These results indicate a lower oxidative capacityof elderly compared with adult mitochondria and

suggest that the loss of muscle oxidativecapacity per muscle volume reflects not onlymitochondrial volume loss but also the reducedcapacity of the mitochondria themselves.


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Adult vs. elderly oxidative capacity

The lower oxidative capacity per mitochondrial volume of the elderlyvastus lateralis compared with the adult group shown in Fig. 6 isconsistent with a lower mitochondrial oxidative enzyme activity withage.

The reduction in mitochondrial function with age may be caused bya number of factors, such as mitochondrial DNA mutatiions,oxidative damage by reactive oxygen species, or reduced synthesisof mitochondrial proteins

Brierly et al. (1997a,b) suggest that the reduction in mitochondrialfunction seen in elderly subjects is not directly caused by an ageingprocess per se.

The end result is decline of nearly half of the muscle oxidativecapacity between adults and elderly subjects due to reducedmitochondrial content as well as a significantly lower oxidative

capacity per mitochondrial volume.


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presentasi biokim ii

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